The Effects of Elevated CO2 Concentrations on Cell Division
Rates, Growth Patterns, and Blade Anatomy in Young Wheat
Plants Are Modulated by Factors Related to Leaf Position,
Vernalization, and Genotype
Josette Masle*
Research School of Biological Sciences, Institute of Advanced Studies, Australian National University,
Canberra, Australian Capital Territory 2601, Australia
Based on experiments at the whole-plant or whole-leaf
level, it has been reported that, relative to other species,
wheat is not very responsive to elevated [CO2], especially
at early stages. It has been argued (Nicolas et al., 1993;
Christ and Ko¨rner, 1995; Slafer and Rawson, 1997) that
while it enhances tillering by promoting the development
of axillary meristems, elevated [CO2] has little to no effect
on leaf development and growth in wheat, a conclusion
also recently put forward for rice, another important cereal
(Jitla et al., 1997). These reports challenged the evidence
from other studies using wheat and a range of other species
showing greatly increased aerial growth rates caused by
elevated [CO2] in very young seedlings, which progressively decrease concurrently with changes in carbon partitioning (e.g. wheat [Neales and Nicholls, 1978; Masle et al.,
1990]; soybean [Rogers et al., 1984]; cotton [Wong et al.,
1992]; tobacco [Masle et al., 1993]). They were also intriguing in the face of the profound developmental effects of
exposure to elevated [CO2] or of Suc feeding recently documented at the cellular and subcellular levels in leaves of
several species, including wheat (e.g. Robertson and Leech,
1995; first leaf of 7-d-old seedlings) and sugar beet (Kovtun
and Daie, 1995; leaves of 4-week-old seedlings).
The aim of the work presented here was to examine the
effects of elevated [CO2] on the spatial and temporal patterns of cell division and cell expansion in developing
wheat leaves and on their translation into variations of
whole-leaf growth kinetics, anatomical features, and carbon content. The analysis was conducted in two genotypes.
Vernalization was used as a way of shifting the timing of
floral initiation and associated changes in carbon allocation
within the plant (e.g. Griffiths and Lyndon, 1985).

This study demonstrates that elevated [CO2] has profound effects
on cell division and expansion in developing wheat (Triticum aestivum L.) leaves and on the quantitative integration of these processes in whole-leaf growth kinetics, anatomy, and carbon content.
The expression of these effects, however, is modified by intrinsic
factors related to genetic makeup and leaf position, and also by
exposure to low vernalizing temperatures at germination. Beyond
these interactions, leaf developmental responses to elevated [CO2]
in wheat share several remarkable features that were conserved
across all leaves examined. Most significantly: (a) the contribution
of [CO2] effects on meristem size and activity in driving differences
in whole-blade growth kinetics and final dimensions; (b) an anisotropy in cellular growth responses to elevated [CO2], with final cell
length and expansion in the paradermal plane being highly conserved, even when the rates and duration of cell elongation were
modified, while cell cross-sectional areas were increased; (c) tissuespecific effects of elevated [CO2], with significant modifications of
mesophyll anatomy, including an increased extension of intercellular air spaces and the formation of, on average, one extra cell layer,
while epidermal anatomy was mostly unaltered. Our results indicate complex developmental regulations of sugar effects in expanding leaves that are subjected to genetic variation and influenced by
environmental cues important in the promotion of floral initiation.
They also provide insights into apparently contradictory and inconsistent conclusions of published CO2 enrichment studies in wheat.

The consequences for plant growth and morphogenesis
of variations in photosynthesis depend on the efficiency of
the conversion of triose-P into Suc and of phloem loading
at the sites of Suc production and unloading in growing
sinks. Ultimately, however, they depend on the sensitivity
to sugar supply of a suite of developmental processes
involved in meristem initiation, cell division, expansion,
and differentiation, and of the mechanisms that regulate
the integration of these processes in the formation of organs with a certain shape, size, and structure. The aim of
the present study was to investigate this latter area using
atmospheric [CO2] as a tool for manipulating sugar supply
to expanding organs and wheat (Triticum aestivum L.) as a
model experimental system.

MATERIALS AND METHODS
Growth Conditions
Wheat (Triticum aestivum L.) plants were grown from
seed in two adjacent, well-ventilated greenhouses matched
for temperature (23°C ⫾ 0.1°C se day and night) and
relative humidity (62% ⫾ 1.0% during the day and 52% ⫾
1.2% at night), but providing contrasted atmospheric CO2
concentrations. One greenhouse was run at present ambient CO2 concentrations, i.e. on a typical day, 350 ⫾ 10 ppm

* E-mail masle@rsbs.anu.edu.au; fax 61–2– 6249 – 4919.
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during the day and 420 ⫾ 19 ppm at night, while the other
greenhouse was run at an elevated CO2 concentration of
900 ⫾ 12 ppm, day and night. Day length increased slowly
over the duration of the experiment (4 weeks) from 11 to
12 h according to seasonal variations in Canberra, Australia
at that time of the year (August to September). The incident
radiation varied from day to day according to outside
conditions around a daily average of 718 ⫾ 40 ␮mol quanta
m⫺2 s⫺1, but was stable over the whole formation of the
leaves used for growth kinematic analysis (see below).
Two wheat cultivars of contrasting genetic background,
morphology, and growth habit were used: cv Hartog, a
selection from the CIMMYT (Centro Internacional de Mejoramiento de Maiz y Trigo) variety Pavon 76, carrier of the
Rht2 dwarf gene and classified as a spring wheat, and cv
Birch 75, a true winter wheat derived from crosses between
English and CIMMYT lines. cv Birch and cv Hartog seeds
were germinated in Petri dishes on wet filter paper and
vernalized at 2°C to 3°C in the dark in a cold room for 7
weeks. By then, seminal roots were 30 to 40 mm long, and
the first leaf 20 to 40 mm long. Five days before sowing,
another batch of seeds was germinated in similar conditions and kept in the dark at 23°C to obtain non-vernalized
“control” seedlings of similar size at sowing as the vernalized ones. All seedlings were transplanted on the same day
to pots filled with a 1:2 sand:perlite mix saturated with
nutrient solution (Hewitt and Smith, 1975). Pots were
flushed once or twice daily with full-strength nutrient solution kept at greenhouse temperature.
Destructive Growth Measurements
Plants of cv Birch and cv Hartog were harvested from
each greenhouse on d 1, 3, 8, d 15 to 17 (date of sampling
for detailed kinematic analysis of leaf elongation, see below), and finally on d 23 (cv Birch) or d 27 (cv Hartog). On
d 3 and 8, only non-vernalized plants were sampled; on the
other dates, both vernalized and non-vernalized plants
were harvested. Roots were cut at the crown level; individual tillers were identified according to their position on
the plant (Masle, 1984) and separated. Leaf blade areas
were immediately measured using an area meter (LI-3000,
LI-COR, Lincoln, NE) and dry weights were determined
after 48 h of oven drying at 80°C. These measurements
were done on each tiller individually, except at the final
harvest, when tillers were bulked according to their biological age (Masle, 1984). The experiment was terminated
when leaf 6 was fully mature (i.e. leaf 8 had just appeared).
Kinematic Analysis of Leaf Elongation
Leaf elongation rates were analyzed non-destructively
through daily measurements of the lengths of all visible
leaves to the nearest 0.5 mm. The underlying cellular responses were investigated using the kinematic approach
pioneered by Goodwin and Stepka (1945) and Erickson and
Sax (1956) for the analysis of axial growth. This method is
based on the principles of fluid dynamics, and treats the
production of cells and their displacement analogously to
those of physical elements within a fluid. It is particularly

Masle

Plant Physiol. Vol. 122, 2000
well suited to cereals and, more generally, grass leaves,
where most of the leaf tissue is generated by a well-defined
basal growth zone made of parallel cellular files whose
expansion is mostly unidirectional and in which division
and elongation occur in two distinct segments. In constant
environments, the blade elongation rate after emergence
from older sheaths is constant (e.g. Friend et al., 1962);
moreover, the spatial distribution of epidermal cell lengths
in the growth zone and the size of that zone (no. of constitutive cells) remain unchanged (data not shown; Schnyder et al., 1990; Beemster et al., 1996). In these “steady-state
conditions,” the spacing of transverse cell walls along the
growth zone can be used to determine cell elongation rates
with respect to both physical position and biological age,
including meristematic cells (Beemster et al., 1996). From
the spatial distribution of newly formed walls in the division zone, local cell partitioning rates can also be derived.

Leaf Elongation Rate, Cell Length Profile, and Size of the
Growth Zone
Leaf 6 was selected for this analysis. Following blade
emergence, leaf length was measured every 3 h over a full
day/night cycle. A regression line was fitted through these
measurements over time (r2 ⬎ 0.999 in all cases), and the
slope of that line was taken as a measurement of the leaf
linear rate of elongation (E). Twenty-four hours after blade
emergence, the leaf was quickly dissected from the plant
under the microscope as close as possible to its insertion on
the apex. The leaf was then immediately immersed in
boiling methanol until all chlorophyll had been removed
and then cleared in lactic acid. Five leaves were analyzed
for each treatment (2 vernalization levels ⫻ 2 CO2 concentrations ⫻ 2 genotypes). Because of [CO2] and vernalization effects on the rate of leaf development (see “Results”),
leaves 6 were not all synchronized and were, depending on
treatment, sampled over 2 d (d 15–17).
The cleared leaf was mounted on a light microscope
(axioscope, Zeiss, Jena, Germany) fitted with a video camera (model WV-CL 702E, Panasonic, Tokyo). A file of epidermal cells of the same type for all leaves (file of sister
cells adjacent to a stomatal row) was selected and individual cell lengths were measured throughout the growth
zone. This was done from video images using the morphometric program MTV (Datacrunch, San Clemente, CA).
Note was taken of the position of thin transverse cell walls,
which are indicative of recent cell divisions. Such walls
were typically visible only in the basal 4 to 7 mm of the
growth zone. The position of the most distal fresh wall was
taken as defining the transition between the division and
the elongation-only zone, where cells had lost the ability to
divide and were starting to undergo rapid expansion. In
many leaves, the last divisions were “asymmetrical,” yielding two daughter cells of very unequal length. The shortest
daughter cells could easily be traced through the elongation zone, where they hardly expanded and started to
differentiate into trichomes. The positions from the base of
the leaf (x0) of the last symmetrical and asymmetrical divisions were denoted as xsd and xad, respectively. Individual cell lengths were also measured along a 10-mm seg-

Elevated [CO2] and Leaf Development: from Cells to Whole Blade
ment of mature blade and their average was taken as an
estimate of final cell length, lf. Individual cell lengths, l(x),
and individual elemental lengths, l(x)* (the length of a cell
and its associated trichome), were plotted against position
(x) along the growth zone. In the elongation-only zone, cell
lengths were fitted by a Richards function as in Morris and
Silk (1992) for cells and Beemster et al. (1996) for elements.
The distal end of the growth zone was defined as the
location (xel) where the fitted cell length reached 95% of lf.
For the meristem, where cell length does not bear any
direct relationship to position, cell length data were
smoothed by calculating moving averages over 11 cell intervals around x. The number of meristematic cells along a
file in the leaf growth zone were denoted as Nsd and Nad
for the zones of symmetrical and asymmetrical divisions,
respectively, and the number of elongating-only cells was
denoted as Nel.

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where n(x) is the total number of cells between xsd and x
and c is the cellochron, the time taken for a cell to be
displaced by one position (Silk et al., 1989).
c⫽

1
F

(6)

and
n共 x兲 ⫽

冕

x

xsd

dx
l共 x兲

(7)

The residence time of a cell in the elongation-only zone,
Tel, was calculated as:
Tel ⫽ c.Nel

(8)

Cell Partitioning Rates
Growth Velocities and Strain Rates
The velocities of displacement v(x) and relative cell elongation rates r(x), the latter often referred to as strain rates in
the kinematic literature, can be calculated as a function of
position x. Velocities in the elongation-only zone are given
by equation 11 in Morris and Silk (1992):
v共 x兲 ⫽ F.l共 x兲

(1)

where F is the cell flux, i.e. the number of cells passing
through x per unit of time, and l(x) are the fitted cell
lengths obtained as described above. During steady
growth, F is constant throughout the elongation zone; the
number of cells displaced out of the meristem into the
elongation zone is equal to the number of cells moving out
of the growth zone and is given by:
F⫽

where ␾(x) is the number of newly formed cross-walls
between the base of the meristem and position x as a
proportion of such walls from the base to xsd (Beemster et
al., 1996).
The relative elongation rates r(x) are the derivatives of
velocities with respect to position (equation 2 in Morris and
Silk, 1992):
r共 x兲 ⫽

dv
dx

(4)

In the elongation zone, time versus position relationships
can easily be calculated. The time, t(x), taken for a cell to be
displaced from xsd (the meristem) to a further particular
position, x, in the elongation zone is given by:
t共 x兲 ⫽ c.n共 x兲

(5)

The average cell partitioning rate, p, in the zone of symmetrical division and its inverse, the average cell cycling
time, tc, time elapsed between two successive divisions,
were calculated as in Green (1976):
p៮ ⫽

F
Nsd

and

t៮c ⫽

Nsd
F

(9)

or
t៮c ⫽ Nsd.c
Local symmetrical partitioning rates at any location x
along the meristem, p(x), were calculated according to the
method of Beemster et al. (1996) as the average partitioning
rate over intervals (i) of m cells around the cell at location
x, using:
p共i, x兲 ⫽

␾共i, x兲
F
m

(10)

where ␾(i,x) denotes the number of newly formed cross
walls found in interval i, as a proportion of the total number of such walls from xo to xsd. Calculations were done on
intervals of 20 cells.
Quantitative Analysis of Mature Blade Anatomy
At final harvest, mature blades of leaf 6 were sampled for
detailed anatomical observations. Four contiguous short
segments, each about 3 mm long (denoted segment 1–4
below), were taken mid-length along the blade.

Morphometric Analysis of Cleared Mature Epidermis
Segment 1 was cleared in methanol and lactic acid as
described above. The numbers of epidermal files between
veins 1 and 2, 2 and 3, and 3 and 4 were counted (veins
numbered from the mid-rib). An 800-␮m-long field of view
delimited by veins 2 and 3, equivalent to 0.2 to 0.3 mm2 of
leaf tissue, was selected on the abaxial side of the blade for
determining the densities and relative proportions of the

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Plant Physiol. Vol. 122, 2000
bers. The slice of fresh tissue was fixed in 3.5% (v/v)
glutaraldehyde for 1 h at room temperature, then transferred into 0.1 m EDTA (pH 9.0), incubated for 3 h at 60°C,
and stored at 4°C. The slice of tissue was later macerated in
5% (w/w) chromium trioxide at 4°C for 24 h until cells
could be easily teased apart without damage. Mesophyll
cell counts were made a few days later using a 0.1-mmdepth hemocytometer under a microscope (Zeiss) on five
loadings per leaf and four leaves per treatment.

various types of cells constituting the mature epidermis
(stomata and interstomatal cells, sister cells in adjacent
rows, elongated non-specialized cells, and trichomes). The
length and maximum width of 10 contiguous cells of each
type were measured, excluding stomata and trichomes.
This was replicated three times per leaf.

Morphometric Analysis of the Mesophyll Tissue on
Leaf Sections
Segments 2 and 3 were immediately fixed in 2% (v/v)
glutaraldehyde in 50 mm 1,4-piperazinediethanesulfonic
acid (PIPES) buffer for 3 h (1 h of which was under vacuum), post-fixed in 1% (v/v) osmium tetroxide in 25 mm
phosphate buffer, pH 7.0, slowly dehydrated in ethanol,
and embedded in Spurr’s resin. Three-micrometer-thick
cross-sections (segment 2) and longitudinal sections (segment 3) were cut, stained with toluidine blue, and mounted
on a microscope fitted with a video camera for morphometric measurements. Cross-sections were used to determine the number of mesophyll cell layers and accurately
measure the thicknesses of the whole blade, mesophyll
tissue, and each epidermis, and the cross-sectional areas of
individual mesophyll and epidermal cells. These parameters were measured at three positions across the blade
centered mid-way between veins 1 and 2, 2 and 3, and 3
and 4. At each position the individual cross-sectional areas
(aj) of all mesophyll cells comprised within a small rectangle of total area A were measured. The difference 1 ⫺
p
兺j⫽1
aj/A was calculated as an estimate of the proportion of
tissue cross-sectional area occupied by air spaces. Depending on treatment, the number of mesophyll cells, p, measured at each location varied from 12 to 20. Four sections
were analyzed at each of three locations across the embedded segment, and there were four replicated leaves per
treatment. Mesophyll cell lengths were measured on 3-␮m
longitudinal sections cut parallel to veins. In wheat, mesophyll cells are elongated with several deep lobes (e.g.
Parker and Ford, 1982), so mesophyll cell lengths were only
measured for cells whose end walls were clearly visible
and abutting the adjacent cells in the file.

Sugar Analyses
Soluble sugars and starch were extracted twice from
dried blade and root powder in 80% (v/v) ethanol. Color
pigments were removed by the addition of activated charcoal (Norit SA3, Aldrich Company, Milwaukee, WI) to the
extract, followed by centrifugation at 12,000 rpm for 10
min. The supernatant was dried down and resuspended in
water. Glc, Fru, and Suc concentrations were determined
enzymatically in three steps after the addition of a formulation of hexokinase and Glc-6-P dehydrogenase (Glc [HK]
diagnostics reagent, Sigma-Aldrich, St. Louis) and of invertase and isomerase. Glc moieties were measured spectrophotometrically at 340 nm following each reaction.
Statistics
Treatment effects on kinematic and morphometric parameters were assessed by analysis of variance using General Linear Models algorithms (statistical package GLIM,
version 3.77, 1985, Royal Statistical Society, London). In
addition, differences in the spatial distributions of strain
rates and partitioning rates were compared by the test of
Kolomgorov-Smirnov.
RESULTS
[CO2] Effects on Whole-Plant Growth, Carbon
Accumulation, and Leaf Expansion
Plants under 900 ppm [CO2] grew significantly more
than those under 350 ppm (Table I), showing a 52% to 93%
increase in total dry weight at the end of the experiment
and a 39% to 82% increase in leaf area. All tillers contributed to that response (Fig. 1), including the main tiller,
where a growth stimulation by elevated [CO2] was detect-

Estimation of Mesophyll Cell Numbers
Segment 4 (segment symmetrical to segment 3 with respect to mid-rib) was used to estimate mesophyll cell num-

Table I. Influence of growth [CO2] and vernalization on whole-plant dry weight and leaf area
(means and SE values) at the end of the experiment in cv Birch (d23) and cv Hartog (d27)
Genotype

Figure 1. Ratio of tiller dry weights under 350 ppm CO2 to tiller dry weights under 900 ppm CO2 at final harvest (d 23 and
27 in cv Birch and cv Hartog, respectively). Labels on the x axis refer to the main tiller (MS) followed by the five first primary
tillers (T1–T5) and tillers of higher order grouped according to biological age (labels 5.1–8.1, corresponding to tillers that
normally emerge concurrently with tiller T3–T7, respectively, and with leaves 6 to 9 on the main tiller, Masle [1984]). White
bars, Ratios for non-vernalized plants; gray bars, ratios for vernalized plants.

able within 4 to 15 d after sowing (Fig. 2) and led to a 25%
to 40% increase in dry weight at final harvest (d 23–27).
Elevated [CO2] accelerated the emergence rate of secondary and tertiary tillers (data not shown), leading to plants
with a greater number of concurrently expanding sinks at
a given time. For those tillers, the size differences shown in
Figure 1 therefore reflect the compounded effects of earlier
appearance and faster growth rate thereafter. Although
elevated [CO2] led to significant increases in the soluble
sugar content in leaves (Fig. 3), this effect was insufficient
to fully account for the increased dry weights shown in
Table I and Figures 1 and 2.
Figure 4 describes the kinetics of elongation of successive
leaves of the main stem, from emergence of the blade to
completion of elongation. In the two genotypes examined,
and whether seeds had been vernalized or not, increased
atmospheric [CO2] stimulated the expansion growth of
individual leaves. Remarkably, however, this stimulatory
effect only became visible and significant beyond leaf 2
(vernalized plants) or even leaf 4 (non-vernalized plants).
Moreover, although in later leaves elevated [CO2] systematically resulted in longer mature blades, the underlying
reasons varied depending on genotype and vernalization
treatment. In vernalized cv Hartog plants, for example,
elevated [CO2] caused earlier blade emergence (see in Fig.
4 the 1–1.5 d lag between the two CO2 levels for leaves 4–8)
and extended growth duration. In the non-vernalized seedlings of the two genotypes, however, these two parameters
were not significantly affected by exposure to elevated
[CO2], but the elongation rate of emerged blades was enhanced. In vernalized cv Birch plants, blades emerged earlier and elongated faster thereafter.

in leaf 6. Table II gives the average blade elongation rate (E)
over the 24-h period following tip blade emergence. For
each individual leaf, E was estimated by the regression line
fitted to the three hourly measurements of blade lengths
taken during that period (see “Materials and Methods”).
Consistent with data shown in Figure 4, E was always
greater under 900 ppm than 350 ppm. Vernalization had no
significant effect except in cv Birch under high [CO2]. Variations in E can be analyzed using Equation 2 in relation to
variations in F, the flux of cells moving out of the growth
zone per unit of time, and in lf*, the length of the newly
fully expanded elements. Table II shows that elevated
[CO2] caused a 15% to 20% increase in cell flux (P ⬍ 5%)
irrespective of genotype and vernalization treatment. In cv
Birch, F was also affected by vernalization, although to a
lesser extent, being 12% greater in non-vernalized than in
vernalized seedlings. In contrast, final elemental lengths
and final cell lengths were remarkably stable across growth
[CO2] levels, vernalization treatments, and genotype (see
Table II; actual overall mean cell length 200 ⫾ 10 ␮m; predicted mean from the analysis of variance ⫽ 199.6 ⫾ 2.8 ␮m).
Elevated [CO2] increased the total elongation rate occurring in both the division zone and the elongation-only zone
distal to it (Esd and Eel, respectively) except in cv Hartog
under vernalization, where Esd was similar in high- and
low-[CO2]-grown leaves (data not shown). The relative
contributions of meristematic and elongating-only cells to
the overall leaf elongation rate, E, were only slightly affected by growth [CO2] or vernalization, varying between
10% to 15% and 90% to 85%, respectively.

Kinematic Analysis of Leaf Elongation

By definition during steady growth the flux of cells
moving out of the growth zone is equal to the number of
cells displaced out of the meristem into the elongation-only
zone during the same period. It therefore integrates variations in both the number of meristematic cells and their

Cell Flux and Mature Cell Length
An analysis of the cellular responses underlying the
effects of elevated [CO2] on leaf elongation was undertaken

Meristem Size and Cell Partitioning Rates

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Plant Physiol. Vol. 122, 2000
The local partitioning rates calculated using the distribution of fresh cell walls (Eq. 10) suggest the existence of a
spatially non-uniform field of responses to elevated [CO2]
along the meristem. As illustrated in Figure 5a, elevated
[CO2] typically enhanced partitioning rates mostly in the
basal 2 to 3 mm of the meristem while having little effect in
the most proximal segment.

Size of the Leaf Growth Zone: Kinetics of Cell Elongation

Figure 2. Main tiller leaf area (top panel) and dry weight (bottom
panel) plotted as a function of time (days from sowing) for the
non-vernalized seedlings of cv Birch and cv Hartog grown under 350
(E) and 900 ppm [CO2] (F); bars across symbols represent 2 SE. Note
that the y axis is common to the two genotypes and is in log-scale.

partitioning rate (Eq. 9). In cv Birch, the average interval
between two successive divisions, tc (calculated using Eq.
9), was 3 to 4 h shorter under 900 compared with 350 ppm
CO2, equivalent to an approximately 13% reduction (Table
III), and the number of symmetrically dividing cells per file
(Nsd) was increased, although relatively less so (⫹8%; P ⬍
0.05) (Table III). In cv Hartog, elevated [CO2] had, qualitatively, similar effects; their relative importance, however,
depended on vernalization treatment (Table III). In vernalized cv Hartog seedlings, faster cycling rates were the
dominant response (14% decrease in tc versus an 8% increase in Nsd), as was also seen in cv Birch, while in the
absence of vernalization, elevated [CO2] had only a weak
effect on tc (5%, non-significant decrease), but caused a 20%
increase in meristematic cell number. Even when individually non-statistically significant, the combined effects of
elevated [CO2] on tc and Nsd always gave rise to highly
significant differences in cell fluxes between low- and high[CO2]-grown plants (Table II).

Elongation in the Meristem. Elevated [CO2] had no significant effect on the distributions of meristematic cell lengths
in either genotype or vernalization treatment (Fig. 6, insets). Variation in meristem length was therefore highly
correlated to variations in meristematic cell number. Consistent with the variations of Nsd described earlier, elevated
[CO2] systematically increased the longitudinal extension
of the division zone (P ⬍ 5%), especially in the nonvernalized seedlings of cv Hartog (Table III, Lsd values).
Elevated [CO2] also enhanced local elongation rates in the
meristem; however, as for partitioning rates, this effect
was mostly confined to the basal 2 to 3 mm of the meristem
(Fig. 5b).
Cell length at division can be estimated from the lengths
of freshly formed daughter cells separated by thin transverse cell walls, and was similar in low- and high-[CO2]grown leaves (20.4 ⫾ 1.8 ␮m). Therefore, elevated [CO2]
caused similar proportional increases in the partitioning
rate and elongation rate of meristematic cells. Figure 5
(example is of cv Birch) illustrates that the distributions of
those two parameters mirrored each other, with the enhancing effect of elevated [CO2] being confined to the basal
half of the meristem.
Elongation of Non-Meristematic Cells. In non-vernalized
seedlings, the length of the elongation-only zone (Lel) and
cell lengths profiles within it and therefore the number of
constitutive cells (Nel) were not significantly affected by
variations in growth [CO2] (Table III; Fig. 6). The temporal
pattern of cell elongation was, however, modified; maximum cell elongation rates were increased (Fig. 7, P ⬍ 5%)
and the duration of cell elongation was significantly shortened (see Tel values in Table III). In contrast, in vernalized
seedlings, high [CO2] increased the length of the elongation
zone and the size of the cohort of concurrently elongating
cells (Nel, Table III), but did not significantly affect maximum cell elongation rates (Fig. 7) nor cell residence times
in the elongation zone (Table III). In cv Birch, however,
elevated [CO2] caused maximum cell elongation rates to
occur relatively further in the elongation zone (Fig. 7) and
later in the expansion time span of the cell (⫹6 h corresponding to a 20% delay compared with cells of low-[CO2]grown leaves) so that within the basal two-thirds of the
elongation zone, cells were shorter than under 350 ppm
CO2 (Fig. 6).
Anatomy of Mature Blades
The above data described growth kinetics of epidermal
sister cells. Morphometric analysis of cleared epidermis
and blade sections allowed examination of the final dimen-

sions of other cell types and of blade anatomy. As found for
sister cells, the lengths of other epidermal cell types (interstomatal cells and non-specialized elongated cells) did not
differ significantly between low- and high-[CO2]-grown
blades (data not shown). The densities of these three cell
types were also similar (Fig. 8), implying that their surface
area and width were also insensitive to ambient [CO2].
Stomatal indices or densities were also not affected (Fig. 8).
The only significant effect of elevated [CO2] was a reduction of trichome frequency in the non-vernalized leaves of
cv Hartog (trichome index reduced from 9.8%–3.9%). Interestingly, vernalization also had little effect on epidermis
anatomy (Fig. 8).
Contrary to our expectations based on observations of
cleared epidermis, the examination of blade sections revealed several striking anatomical differences between leaf
tissue generated under 350 or 900 ppm CO2, which also
depended on both genotype and vernalization. While consistently having no effect on mesophyll cell length or cell
projected area (Table IV), elevated [CO2] caused a significant increase in the cross-sectional areas of those cells
except in the cv Hartog non-vernalized seedlings (Fig. 9,
top panel). No such increase was observed in epidermal
cells except in the non-vernalized leaves of cv Birch, where
the cross-sectional area of epidermal cells of all types was
also greater in high-[CO2]-grown leaves (Fig. 9, bottom
panel). Remarkably, elevated [CO2] consistently caused an
increase in air space volume in the mesophyll tissue. This
latter effect was more marked in cv Hartog, and significant
at any position across the blade (Fig. 10). In cv Birch, it was
closest to the mid-vein while disappearing toward the

blade margins. In addition, elevated [CO2] caused an increase in the number of mesophyll cell layers (on average,
one out of three to five layers in total, data not shown) with
the exception, again, of the non-vernalized seedlings of cv
Hartog. Overall, these various effects resulted in an increase in blade thickness and structural carbon content per
unit leaf area in high-[CO2]-grown leaves (Fig. 10); in the
non-vernalized leaves of cv Hartog, however, increased
leaf thickness was confined to the mid-rib region. Because
ambient [CO2] had no effect on the size of major or minor
veins (vein diameters of 60–80 ␮m regardless of [CO2] and
vernalization, data not shown), the ratio of mesophyll tissue volume to vein volume was greater in high-[CO2]grown leaves.

DISCUSSION
Elevated [CO2] Caused an Early Stimulation of Plant
Growth and Modified the Development of
Individual Leaves
Contrary to earlier conclusions in the literature, wheat
proved to be an excellent model system to analyze [CO2]
effects on leaf development. In the two genotypes examined, vegetative growth was increased under 900 ppm CO2.
The stimulatory effect of elevated [CO2] was detectable
early, in the first week after sowing, on leaf area expansion
and biomass accumulation, at a time that did not coincide
with any specific phenological stage nor necessarily with
the beginning of tillering (Fig. 4). Depending on genotype
and vernalization, a positive growth response to elevated

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Plant Physiol. Vol. 122, 2000

Figure 4. Comparison of blade elongation for successive leaves of the main tiller (leaf 2–8) under 350 (dashed line) and 900
ppm CO2 (solid line) in vernalized (left) and non-vernalized (right) seedlings of wheat cv Birch and cv Hartog. Blade length
was measured from the ligule of the subtending leaf. Arrows on the x axis denote the times of first tiller emergence in lowand high-[CO2]-grown plants (thin and thick arrows, respectively).

[CO2] was detected before the appearance of the first tiller
(cv Hartog, vernalized leaves), at approximately the same
time (e.g. cv Birch, vernalized leaves), or significantly later
(e.g. Birch non-vernalized leaves) (Fig. 4). One can therefore conclude that there is no direct causal link between the
two events, as has been suggested (e.g. Nicolas et al., 1993;
Christ and Ko¨rner, 1995). This study demonstrates pro-

found effects of elevated [CO2] on individual leaves of the
main tiller, even before axillary meristems become major
competing sinks for carbon. As will be discussed below,
these effects are both quantitative and qualitative.
Within a few days after the effects of high [CO2] on leaf
growth were detected, high-[CO2]-grown plants were characterized by greater ratios of carbon (total or structural)

Table II. Influence of growth [CO2] and seed vernalization on leaf elongation rate (E), the lengths of
mature sister cells or elements (If and I *f , respectively), and the cell flux (F, number of cells and elements moving in and out of the leaf epidermal elongation zone per unit of time)
[CO2] and vernalization had statistically significant effects (P ⱕ 0.05) on E and F (values followed by
a different letter within a column) but not on If and I*f (GLM analysis of variance, see “Materials and
Methods”).
Genotype

Birch

Vernalization

Yes
No

Hartog

Yes
No

[CO2]

E

ppm

mm/h

350
900
350
900
350
900
350
900

1.45a
1.70cd
1.54ab
1.89e
1.53ab
1.76d
1.63bc
1.78d

I *f

If

cells h⫺1

␮m

218
208
206
205
210
198
206
195

F

210
201
201
203
201
189
200
190

6.7a
8.2cd
7.5c
9.2e
7.4b
8.9d
8.0b
9.2d

Elevated [CO2] and Leaf Development: from Cells to Whole Blade

1407

Table III. Influence of growth [CO2] and vernalization on the characterictics of the leaf growth zone
during the phase of steady elongation following blade tip emergence
The three parameters given for the division zone and the elongation-only zone refer to: the physical
extension of these zones (Lsd and Lel), the number of constitutive cells (Nsd and Nel), the average cell
cycling time, ៮tc , for meristematic cells or residence time in the growth zone (Tel) for elongating-only
cells. Values followed by a different letter within a column were statistically significantly different (P ⱕ
0.05).
Genotype

Birch

Vernalization

Yes
No

Hartog

Yes
No

[CO2]

Division Zone

Elongation-Only Zone

Nsd

tc

Lsd

Nel

Tel

Lel

ppm

cells

h

mm

cells

h

mm

350
900
350
900
350
900
350
900

172a
186a
226b
244bc
268cd
290d
233b
280d

26.1b
22.9a
30.5cd
26.5b
36.9e
32.6d
29.5bc
27.8a

3.9a
4.4b
5.0c
5.7d
6.8e
7.3f
5.7d
6.6e

248a
313b
266a
257a
301b
365c
296b
290b

37.6cd
38.1cd
35.4c
27.8a
41.4d
41.2d
37.3cd
32.0b

22.7a
27.6bc
23.7a
23.4a
29.6c
33.1d
26.8b
26.0b

Figure 5. Local cell partitioning rates (a) and relative elongation rates
(b) averaged over successive cohorts of 20 cells along the growth
zone under 350 and 900 ppm [CO2] (dashed and solid lines, respectively; vertical bars ⫽ 2 SE). Data are for leaf 6 of vernalized cv Birch
seedlings; [CO2] effects followed similar patterns in non-vernalized
leaves and also in cv Hartog. On the x axis, positions along the
growth zones are described by the distance from the distal end of the
division zone, xsd.

laid down in the whole plant to leaf area expanded. These
differences mostly reflected an increase in C content per
unit leaf area, a feature observed in other CO2 enrichment
studies with wheat or other species and, more generally, in
response to increased CO2 assimilation rates (e.g. Thomas
and Harvey, 1983; Vu et al., 1989; Masle et al., 1993). The
relative allocation of carbon between roots and shoot was
little affected by growth [CO2]. Higher carbon contents per
unit leaf area in high-[CO2]-grown leaves have been observed in other studies and interpreted as merely reflecting
a more advanced stage of development in these leaves, i.e.
an ontogenetic drift (see also the “temporal shift model”
proposed by Miller et al. [1997]; Sims et al., 1998). In the
present study, homologous leaves were characterized by
different carbon densities depending on growth [CO2] not
only while expanding, but also when fully mature (Fig. 10).
Our data leave no doubt that there are intrinsic developmental differences between low- and high-[CO2]-grown
leaves, which overall lead to the deposition of more structural carbon per unit leaf area in the latter. Elevated [CO2]
affected cell division and expansion rates (Figs. 5 and 7)
and also leaf histogenesis, final dimensions, and anatomy
(Figs. 9 and 10). Furthermore, for the first time to our
knowledge, it is shown that these quantitative and qualitative effects of elevated [CO2] on leaf growth are subject to
significant intraspecific genetic variation and, most unexpectedly, are modified by seed vernalization, a major developmental cue for the switch between vegetative and
reproductive development in cereals.
The effects of atmospheric [CO2] that are reported here
were primarily due to increased photoassimilate supply
(see in Fig. 3 the increased sugar contents and the 25%
increase in rate of leaf photosynthesis per unit leaf area
measured in a preliminary experiment and similar growth
conditions). Elevated [CO2] caused a reduction in stomatal
aperture, but, due to the well-ventilated conditions of the
greenhouses, there was no detectable difference in leaf
temperature between low- and high-[CO2]-grown plants.
Although one cannot totally exclude some role for improved leaf water status under elevated [CO2], this effect

1408

Masle

Plant Physiol. Vol. 122, 2000

Figure 6. Cell lengths (means and corresponding SE) as a function of position along the growth zone in leaf 6 of vernalized
(left) and non-vernalized (right) seedlings of cv Birch and cv Hartog grown under 350 (E) or 900 ppm CO2 (F). Cell lengths
at the base of the growth zones (in the meristem and proximal end of the elongation-only zone) are shown in more detail
in the insets. The curves were obtained by fitting the data using a Richards function, as described in “Materials and
Methods.” Arrows on the x axis denote the position xel, where cell lengths were within 5% of mature cell length under 350
and 900 ppm CO2 (thin and thick arrows, respectively).

was most likely minor. The soil was maintained very wet
and, more importantly, quantitatively similar positive responses to elevated [CO2] as those described in Figures 1
and 4 were observed in a parallel experiment where plants
were grown in hydroponics and under higher air humidity
(6–7 mbar leaf-to-air vapor pressure difference against
11–14 mbar in this experiment [J. Masle, unpublished
data]).

Cellular Bases of [CO2] Effects on Leaf Growth and
Carbon Deposition per Unit Leaf Area
The cellular responses underlying whole-leaf responses
to elevated [CO2] were examined using a combination of
microscopy techniques and the theoretical framework developed earlier for the kinematic analysis of “steady-state”
growth zones, characterized by constant cell length profiles
(see “Materials and Methods”). All leaves analyzed in the
present study were sampled at similar developmental
stages during the period of linear elongation that followed
blade emergence. The ligule meristem was then just initiated and at 0.5 mm at the most from the base of the leaf, so
that the whole growth zone could be treated as one continuous zone for the derivation of kinematic parameters

(Kemp, 1980; Schnyder et al., 1990; Skinner and Nelson,
1995).

Elevated [CO2] Enhanced (Epidermal) Cell Division Rates
A consistent effect of elevated [CO2] was to reduce the
time interval between successive divisions (Table III). This
is the first time that such an effect has been demonstrated
in expanding leaves. Earlier studies gave evidence for a
direct role of Suc in mitosis in both buds (Ballard and
Wildman, 1964) and root meristems (Webster and Henry,
1987), but being based on comparisons of mitotic indices,
these data did not allow the separation of the respective
contributions of changes in the cell cycle per se versus
changes in the number of cycling cells. The present finding
of faster cycling rates in leaves expanding under elevated
[CO2] is consistent with the recent report by Kinsman et al.
(1997) of enhanced cell division rates in the shoot apex of
Dactylis under 700 ppm CO2 compared with 350 ppm.
Furthermore, although determined by different methods,
their estimates of cell cycling times are comparable to ours,
however, with a more pronounced CO2 effect (20% change
for a doubling in atmospheric [CO2]).

Elevated [CO2] Affects Epidermal Cell Elongation Rate But
Not Cell Length at Partitioning Nor Final Cell Length
Elevated [CO2] also affected cellular growth in the meristem and beyond. This study reveals several new features
about these effects. Growth [CO2] influenced local rates of
cell elongation but, remarkably, had no detectable effect on
cell length at partitioning (lsd), i.e. upon cell entry into the
elongation-only zone (Fig. 6). Nor did it affect mature cell
length (lf, Table II) or width (data not shown). The elongation rates of meristematic cells were consistently increased
by elevated [CO2]. In non-vernalized leaves this stimulatory effect of elevated [CO2] on wall elongation was maintained after cell migration out of the meristem (greater
rmax, Fig. 7). In those leaves non-meristematic cells elongated faster under elevated [CO2] but for a shorter time
(Table III), with the net result being no change in lf. In
vernalized leaves, however, none of these effects of elevated [CO2] was detectable. The maximum rate of cell
elongation and the cell residence time in the elongationonly zone were insensitive to [CO2] (Fig. 7; Table III), hence
the conserved final cell length.
There are two possible hypotheses for the constancy of
lsd and lf. The first hypothesis is that for cell partitioning to
occur, meristematic cells have to reach a set threshold size
and, once they have lost the ability to divide, cells elongate
to a fixed length. Under that interpretation there is no
direct sugar effect on cell cycling time or duration of cell
elongation. Variations in tc and Tel with growth [CO2]
follow from [CO2] effects on cell elongation rates. In the
absence of such effects, as in vernalized leaves (Fig. 7), Tel
is unchanged. This first interpretation would require mech-

Table IV. Influence of growth [CO2] and vernalization on the
length and projected area of mature mesophyll cells
Genotype

Birch

Vernalization

Yes
No

Hartog

Yes
No

[CO2]

Mesophyll Cell
Length

Projected area

ppm

␮m

␮m2

350
900
350
900
350
900
350
900

171a
167a
163a
160a
122b
109b
126b
137b

3058b
3125bc
I
I
2746a
2812ab
3316c
3430c

anisms by which cells can measure their size. While there is
some evidence for that in yeast (e.g. Nurse and Fantes,
1981), it is totally unknown whether such mechanisms
operate in higher plants (Francis and Halford, 1995). Data
from our other experiments (Beemster et al., 1996) have
shown that there is no absolute size threshold for cell
division in wheat, and no stable relationship between the
partitioning rate and the elongation rate in meristematic
cells.
The second hypothesis is that cell partitioning and cessation of growth are determined by the cell metabolic
status and/or state of differentiation rather than by size per
se. Under that interpretation, the concurrent decrease in tc
(or Tel) and increase in rsd (or rel) observed under elevated
[CO2] are caused by the increased metabolic activity associated with increased photoassimilate supply. The constancy of lsd and lf seen in this experiment would then
simply mean that the rate of cell elongation and the rate at
which a cell reaches the “critical” state, conditioning partitioning and cessation of growth, are increased by a similar proportion when cellular sugar concentrations increase. However, there is no reason to exclude the
possibility that in other species and under different growth
conditions, these two sets of parameters may show a differential sensitivity to sugars, leading to variations in lsd or
lf as reported, for example, by Ferris et al. (1996) in perennial rye-grass under elevated [CO2] or by Beemster et al.
(1996) in wheat under root stress. This second interpretation for the constancy of lf in this experiment appeals to us
in that it provides a unified explanation for the present
data as well as others where lsd and/or lf have been found
to vary. Furthermore, while not requiring any role of cell
size per se it does not exclude it.
Whether direct or indirect a tight link between [CO2]
effects on cell division and expansion processes is shown
by the correlation between the spatial patterns of local rates
of cell partitioning and cell elongation (Fig. 5). Furthermore, the fact that [CO2] effects on cell elongation are most
important at the base of the meristem (Fig. 5) and smaller
in the elongation zone indicates that these effects are modulated by factors related to cell position and/or meristematic status. Increased cellular Suc contents in the basal part
of the growth zone may be one of these factors, as suggested by Schnyder and Nelson (1987) data in Festuca.

Plant Physiol. Vol. 122, 2000

Elevated [CO2] Modifies Growth Anisotropy
Morphometric analysis of mature blade sections demonstrates that elevated [CO2] has more complex effects on
cellular growth than revealed by the examination of cell
lengths and does modify cell properties. Thus in cv Birch,
while not affecting final cell lengths (Tables II and IV),
elevated [CO2] caused a significant increase in the crosssectional area of mesophyll cells and, in non-vernalized
leaves, also of epidermal cells (Fig. 9). In cv Hartog,
changes in mesophyll cell cross-sectional area were only
observed in vernalized leaves. These observations lead to
the suggestion that elevated [CO2] modifies the anisotropy
of cell growth, and that the underlying control mechanisms
are genetically variable and sensitive to factors influenced
by exposure to low vernalizing temperatures. Moreover,
the absence of a stable correlation between dimensional
changes observed in epidermal and mesophyll cells (see
Fig. 9) indicates that [CO2] effects on growth anisotropy
may be in part tissue or even cell type specific.

Figure 9. Mesophyll cell cross-sectional area (top panel; means and
SE) and epidermal sister cells cross-sectional area (bottom panel)
calculated as described in “Materials and Methods” in mature leaves
grown under 350 (white bars) or 900 ppm CO2 (gray bars). Data
(means and SE) are shown for the two cultivars (left, cv Birch, and
right, cv Hartog), and for vernalized and non-vernalized leaves (label
v and nv on the x axis, respectively).

Elevated [CO2] and Leaf Development: from Cells to Whole Blade

1411

Figure 10. Decrease in blade thickness from the mid-rib to the edge of the blade. Measurements were taken on thin
cross-sections across the mid-rib (MR) and the four adjacent veins (veins numbered 1–4 from MR) in mature leaves (leaf 6)
grown under 350 or 900 ppm CO2 (white and black symbols, respectively). Bars across symbols denote SE. The two rows
of values above the x axis describe the extent of air spaces as a proportion of mesophyll tissue at the same locations (see
“Materials and Methods”), with bold values (first row) referring to high-[CO2]-grown leaves and the values below referring
to leaves grown under 350 ppm CO2. Boxed values are the averages across all positions. In the top right corner of each
panel, average structural carbon contents (mol m⫺2) are given for high- and low-[CO2]-grown blades (in bold and normal
characters, respectively). Left, Vernalized seedlings; right, non-vernalized seedlings.

From Cells to Whole Leaf (Regulation of Whole-Leaf
Elongation Rate)

The Enhancement of Leaf Growth Rate under Elevated
[CO2]: a Crucial Role of Effects in the Meristem
The kinematic methods of growth analysis provide a
conceptual and mathematical framework for a quantitative
analysis of the relationships between cell and whole-organ
responses (Erickson and Sax, 1956; Green, 1976; Gandar,
1983a, 1983b) from two points of view:
First, whole leaf elongation, E, can be expressed as the
integral of local strain rates, r(x), over the length of the
whole growth zone, with no explicit role of partitioning
rate nor of the number of cells contributing to growth. This
approach (Fig. 7) indicates that differences in E with
growth [CO2] arose in part from differences in the spatial
regulation of cell wall elongation along the growth zone,

with elevated [CO2] either up-regulating r at a given position without affecting the size of the growth zone (nonvernalized leaves), or displacing the peak of maximum
cellular activity toward more distal positions along a
longer growth zone (vernalized leaves).
Second, leaf elongation may also be seen as the integrals
of growth activities of a certain number of cells, which
sequentially move away from the meristem. Attention is
now focused on the growth trajectories of individual material particles (cells here) with respect to time in considering that the endowment of a cell for elongation may be
specified at the outset (e.g. see Van’t Hof, 1973, and discussion in previous section) and that the size of the growth
zone may be related to cell number rather than being
physically fixed. This second approach, encapsulated in
Equation 2, points to the importance in the present experiment of meristem size and activity in determining varia-

1412
tions in leaf growth and anatomy with growth [CO2]. With
lsd and lf being highly conserved, variations in the overall
elongations generated in the division and elongation-only
zones were directly proportional to variations in cell flux,
F. Furthermore, variations in final leaf length were proportional to those in total number of cells per file, i.e. in the
number of proliferative divisions.
The importance of cell flux, often referred to as the cell
production rate, in driving environmental effects on leaf
expansion has been emphasized in several recent studies
on roots (e.g. Muller et al., 1998), all of which concluded
with the dominant role of changes in the number of cycling
cells (Nsd) rather than changes in cell cycling rate (tc). This
cannot be generalized to the [CO2]-induced increase in
epidermal cell fluxes observed in this study. The present
data demonstrate that elevated [CO2] may affect both Nsd
and tc and that its relative impact on each parameter varies
depending on genotype and vernalization treatment.

A Relative Insensitivity of Leaf Area Expansion to Elevated
[CO2]: CO2 Effects on Leaf Anatomy
In all cases, at the leaf level, elevated [CO2] had relatively
less effect on processes involved in expansive growth than
on those determining growth in mass, leading to increased
structural carbon content per unit leaf area (Fig. 10). This
increase was not necessarily associated with increased
blade thickness (e.g. in cv Hartog, non-vernalized leaves), a
common characteristic of high-[CO2]-grown leaves (e.g.
Downton et al., 1980; Thomas and Harvey, 1983). Increased
carbon content was the overall result of, first, a large insensitivity of the final structure of the epidermisâ&#x20AC;&#x201D;a tissue
thought to place major constraints on expansive growth in
leaves (Kutschera et al., 1987)â&#x20AC;&#x201D;to sugar supply. This insensitivity is manifest in the highly conserved epidermis
anatomy, including stomatal density and cell dimensions
in the paradermal leaf plane. Increased carbon content
was also due to complex histological changes in the mesophyll tissue affecting both mesophyll cell initiation and
development.
While the formation of an extra mesophyll cell layer
under elevated [CO2] or an increase in mesophyll cell
enlargement have been noted in other C3 species (soybean,
Thomas and Harvey, 1983; Phaseolus, Radoglou and Jarvis,
1992), the systematic increase in intercellular air spaces in
high-[CO2]-grown leaves is an intriguing new finding. The
formation of air spaces in leaves is generally thought to be
developmentally regulated and to occur in a predictable
manner involving localized lysis of cell wall components
and local cell wall thickening (Knox, 1992; Raven, 1996). Its
sensitivity to atmospheric [CO2] shown here is, however,
consistent with experimental evidence and theoretical prediction that cell separation forces increase with cell turgor
and cell diameter (Jarvis, 1998). Indeed, Suc contents were
significantly greater in the high-[CO2]-grown leaves (Fig.
3), most likely resulting in increased mesophyll cell osmotic pressure and turgor; furthermore, in both cv Birch
and cv Hartog, mesophyll cell diameter was increased.
While the formation of more numerous mesophyll cell
layers under elevated [CO2] would allow increased carbon

Masle

Plant Physiol. Vol. 122, 2000
deposition per unit leaf area, the greater extension of intercellular spaces plays in the opposite direction. The fact
that leaf C/m2 was increased even when this latter effect
was the largest or even the only one to be significant, as in
cv Hartog (non-vernalized leaves), implies that in these
leaves, elevated [CO2] either increased carbon/unit cell
wall area and/or increased the mesophyll cell area to volume ratio, through changes in cell lobing for example.
The significance of the anatomical changes induced in
wheat leaves by elevated [CO2] for leaf function needs to
be considered. Effects that contribute to increased leaf
carbon/m2 constitute limitations to the magnitude of longterm whole-plant growth enhancement by elevated [CO2]
(see equation 1 in Masle et al., 1990). On the other hand,
more developed air spaces and increased cell area to volume ratio should facilitate CO2 diffusion in the mesophyll
tissue (Parkhurst, 1994), resulting in a smaller drop of CO2
partial pressure between the substomatal cavity and sites
of carboxylation, and thereby in higher effective assimilation rate at a given stomatal conductance.
[CO2] Effects on Leaf Growth Are Modified by Factors
Related to Leaf Position, Vernalization, and Genotype

Genotypic Effects
There have been several recent reports of interspecific
variation in growth responses to elevated [CO2] within a
genus (Populus, Gardner et al., 1995; Dactylis, Kinsman et
al., 1997). This study demonstrates intraspecific variation
among cultivars of an intensively bred species. Genetic
variation is shown in both the magnitude of [CO2] effects
on a range of developmental processes and in the relative
contributions of these processes to variations in leaf growth
rate and anatomy. Our data, however, also reveal some
highly conserved attributes, such as final cell length or
number of epidermal cell files, which constrained leaf
growth responses to elevated [CO2] in the two genotypes
examined. These two groups of attributes define targets for
an effective genetic manipulation of growth responses to
[CO2] by classical breeding programs using natural genetic
variation and by more directed genetic engineering.

Vernalization Effects
Aside from modifying the effects of atmospheric [CO2]
on the size of the growth zone and on the kinetics of
elongation within it, vernalizing temperatures had profound effects per se on overall blade elongation rate and
mature blade anatomy. Unexpectedly, this was the case
even in a genotype such as cv Hartog, classified as a spring
type based on its vernalization requirements for flowering.
In many respects, however, the effects of seed vernalization
differed between cv Hartog and cv Birch, especially in the
meristem. In the winter vernalized cv Birch leaves were
characterized by a smaller meristem, comprising fewer
epidermal cells with a faster partitioning rate than in nonvernalized seedlings (Table III) and smaller mesophyll cells
(Table IV). In the spring cv Hartog, the opposite effects
were observed, i.e. a longer meristem with more numerous

Elevated [CO2] and Leaf Development: from Cells to Whole Blade
epidermal cells that cycled more slowly and thicker mesophyll cells. In the two genotypes, however, the two sets of
effects resulted in only a small change in cell flux of similar
direction (10% greater flux in non-vernalized seedlings).
Vernalization systematically increased the residence time
of cells in the elongation zone, especially under elevated
[CO2] (Table III), but caused a decrease in cell elongation
rates (Fig. 7) so that final cell length remained mostly
unchanged (Table II).
Overall, these data reveal that early exposure to low
vernalizing temperatures affects the expression of a number of genes involved in leaf development and that these
effects are modified by the plant carbohydrate status.
Whether there is a direct link between the observed effects
of low temperatures on leaf development and on vernalization per se, i.e. on the promotion of flowering, or
whether these are independent effects is an open question.
Several decades ago, Purvis and Hatcher (1959) observed in
several cereals, including wheat, that vernalized seedlings
had a shorter coleoptile and first leaf than non-vernalized
ones. Since then, several genes controlling vernalization
requirements in wheat have been identified (vrn genes),
but studies of their expression have been restricted to a few
genotypes with respect to flowering response and apical
development only, with no attention being paid to possible
pleiotropic effects on leaf development.

Leaf Position Effect
A feature common to all [CO2] growth responses analyzed in this experiment is that significant [CO2] effects on
leaf elongation could not be detected in the first two to four
leaves. A similar pattern was noted by Williams and Williams (1968) in their analysis of expansive growth under
different light levels, and is also present in the data of
Friend et al. (1962), which also showed variation in irradiance. How can this be explained? While it may be argued,
following Williams (1960), that leaves 1 and 2 derive most
of their carbon from seed reserves rather than from current
photosynthesis, this is not the case for subsequent leaves.
By the time leaf 3 emerges, seed reserves are depleted.
Furthermore, as discussed earlier, the data presented in
Figure 4 also rule out a direct causal relationship between
the onset of tillering, which causes a sharp increase in
carbon demand by axillary meristems and the appearance
of a carbon limitation in the expanding leaves of the main
tiller.
A third, non-exclusive interpretation is that the sensitivity of leaf growth to carbohydrates is developmentally
regulated and is confined to early stages in leaf ontogeny.
Recent molecular studies provide several examples of
genes that are differentially regulated by sugars depending
on the physiological/developmental context of the leaf
(Kovtun and Daie, 1995). In the present experiments, leaves
1 to 5 all started to develop before sowing, i.e. before first
exposure to different ambient [CO2]. In wheat, leaves 1 to
3 are initiated in the embryo of the maturing seed, where
their development is arrested by seed desiccation three to
one plastochrons after initiation, respectively (Williams,
1960). Their development resumes slowly upon seed imbi-

1413

bition and, in this experiment, during the subsequent period preceding sowing (see “Materials and Methods”).
Over that period, which in thermal time was about 120
degree.days long (48 d at 2.5°C), two additional leaves
were initiated (leaf 4, and just before sowing leaf 5, data not
shown). On that basis we propose that the effects of sugars
on the wheat leaf growth and final anatomy identified here
may be largely determined in the leaf primordium, during
the first three to five plastochrons of its development. Our
recent observations on leaf development in relation to variations in root environment suggest a simple explanation
for that. In a study on the effects of root impedance on leaf
expansion in wheat (Masle, 1998), we found that a step
change in soil strength only modified expansion growth
and final leaf dimensions of those leaves which, upon
imposition of the step change, were still enclosed in the
whorl of older sheaths, i.e. were four to five plastochrons
old at the most. We concluded that the kinetics of blade
elongation and many attributes of the adult leaves were
determined at these early stages. This explanation alone
would lead one to expect, as observed in the present experiments, no to small CO2 effects on all leaves initiated
before exposure to high [CO2], the more so in the older
leaves. It would also account for the results of recently
published studies where step changes in [CO2] had no
effect on the elongation rate of currently expanding leaves
(e.g. Christ and Ko¨rner, 1995), leading to the erroneous
conclusion that in wheat leaf growth per se is not carbon
limited.
The data presented here demonstrate that elevated [CO2]
has profound effects on leaf development, expansive
growth, and anatomy in wheat. They reveal that these
effects are modulated by intrinsic factors related to genetic
makeup and to leaf position, and by environmental cues
important in apical development. They also identify important developmental constraints to the magnitude of
whole-leaf responses to increased photoassimilate supply
related to the controls of mature cell length, cell growth
anisotropy, and number of cellular files contributing to
lateral blade expansion. Similar types of interactions and
limitations are likely to operate in many species (see the
leaf position effect on expansion responses to elevated
[CO2] noted in bean by Leadley and Reynolds [1989]; see
also the interactions between [CO2] and photoperiodic requirements for flowering reported by Kinsman et al. [1997]
in Dactylis). Their understanding holds the key to realistic
predictions of vegetation (or even single species) responses
to rising [CO2] levels. It is also essential for the design of
appropriate strategies for the engineering of genotypes
most able to maximize the benefits of higher atmospheric
[CO2] in a given environment. More fundamentally, the
elucidation of the functional bases of the novel interactions
identified in this study is essential for understanding the
role sugars play in the regulation of developmental genes
under natural growth conditions.
ACKNOWLEDGMENTS
I thank Joanna Maleszka and Jason Chapman for superb technical assistance, Chin Wong and Peter Groeneveld for their work

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in the design and computer control of the greenhouses, and Sandra
Lavorel for her advice with statistical analysis of data.
Received August 19, 1999; accepted December 7, 1999.